Summary: Herpes simplex virus-1 (HSV-1) infects and replicates in a broad range of cell types and is thus associated with a wide range of disease. Within the nervous system, HSV-1 commonly establishes a latent infection in peripheral neurons. This active infection is suppressed by an immunocompetent host immune response. However, HSV infection of the central nervous system (CNS) can cause herpes simplex encephalitis that, especially in immune-impaired individuals, results in death or severe and long-lasting neurological sequelae. Replication-competent neuroattenuated HSV-1 vectors-- currently used in clinical trials for the treatment of cancers including brain tumors -- are reported to replicate poorly in the CNS. This research program focuses on 1) the ability of these and other HSV vectors to replicate productively in normal cell types in vivo, 2) the cellular and viral factors important for robust productive replication of these vectors, and 3) the effect of infection and associated immune response on neuronal function. In the last year, this program has identified one normal cell type, ependymal cells, in the murine CNS that supports, in vivo, the productive replication of HSV vectors deleted in ICP34.5. The reason why these viruses replicate in this normal cell type will be explored. Studies focusing on cellular factors involved in viral replication and pathogenesis have progressed as well. During late stages of cellular infection by HSV, a nuclear structure is formed that contains at least 4 viral proteins: UL3, UL4, UL20.5 and ICP22/Us1.5. Of these four proteins, the function of only ICP22/Us1.5 is in part understood. Importantly, ICP22 is thought to be involved in neurovirulence; the lethal dose (LD50) by intracranial inoculation of mice with viruses deleted in ICP22 is much higher than with wild type virus. In the last year, several roadblocks to identifying the cellular factors associated with these nuclear structures have been removed. Mistakes in the literature have been identified and corrected; the transcript responsible for translation of UL3 has been identified. Together with mutagenesis studies, these data suggest that the UL3 ORF is smaller than expected. Previously a cellular protein was identified to interact with a larger and thus incorrect form of UL3 in the yeast two-hybrid system. These recent findings allow us now to determine whether this cellular protein associates with the UL3 present in infected cells.